The present invention relates to nucleic acids encoding human glycine transporter type 2 (GlyT2) molecules, to proteins encoded by such nucleic acids, to methods of characterizing GlyT2-active compounds, to uses of DNA and RNA nucleotide probes directed to nucleic acids encoding the GlyT2 transporter, to uses of antisense molecules to inhibit GlyT2 expression, to uses of the GlyT2 protein to generate GlyT2-specific antibodies, and to screening methods using GlyT2-expressing cell lines, and to the field of drug discovery.
The termination of synaptic transmission in the central nervous system (CNS) involves either the enzymatic inactivation of neurotransmitters, or their uptake into pre-synaptic terminals or surrounding glial cells (Amara, S. G. and Kuhar, M. J., Annu. Rev. Neurosci. 16:73-93 (1998); Malandro, M. S. and Kilberg, M. S., Annu. Rev. Biochem. 65:305-336 (1996)). High-affinity, membrane-associated transporters typically mediate the rapid removal of neurotransmitters from the synaptic cleft, with uptake across a concentration gradient being thermodynamically coupled to transmembrane ion gradients (Kanner, B. I., Curr. Opin. Cell Biol. 1:735-738 (1989)).
Neurotransmitter transporters can be separated into two structurally-distinct families. One family mediates Na30-dependent excitatory amino acid uptake (Kanai, Y. and Hediger, M. A., Nature 360:467-471 (1992); Pines, G., et al., Nature 360:464-466 (1993); Storck, et al., Proc. Natl. Acad. Sci. (USA) 89:10855-10859 (1992)). The other family contains several members involved in the Na30/Clxe2x88x92-dependent transport of a host of other neurotransmitters, including GABA (Borden, L. A., et al., J. Biol. Chem. 267:21096-21104 (1992)); catecholamines (Pacholczyk, T., et al., Nature 350:350-354 (1991)); glycine (Kim, et al., Mol. Pharm. 45:608-17 (1994); Liu, Q. R., et al., FEBS Lett. 305:110-114 (1992a)); proline (Fremeau, R. T., et al., Neuron 8:915-926 (1992)) and taurine (Liu, Q. R., et al., Proc. Natl. Acad. Sci. (USA) 89:12145-12149 (1992b)). The gene products for most of the latter family have recently been cloned and sequenced. These transporters demonstrate 40-50% amino acid similarity and likely exhibit structural conservation, as membrane topology analysis predicts twelve putative transmembrane regions (Guastella, J., et al., Science 249:1303-1306 (1990); Smith, K. E., et al., Neuron 8:927-935 (1992)). The high homology within these transmembrane regions has facilitated the design of degenerate primers for the cloning of additional members of the transporter superfamily using PCR-based technologies (Borowsky, B., et al., Neuron 10:851-863 (1993); Hoffman, B. J., et al., Science 254:579-580 (1991)).
Glycine is a major inhibitory neurotransmitter in the spinal cord, brainstem and retina, where it exerts its effects on the strychnine-sensitive glycine receptors (Betz, H., et al., Ann. N. Y. Acad. Sci., 207:109-115 (1993); Betz, H., et al., Q. Rev. Biophys. 25:381-394 (1992)). In addition, glycine acts as a co-agonist with glutamate at the NMDA receptor (Kemp, J. A. and Leeson, P. D., Trends. Pharmacol. Sci. 14:20-25 (1993); Benveniste, M., et al., J. Physiol. (London) 428:333-357 (1990)). Synaptic glycine concentrations are controlled by Na+/Clxe2x88x92-dependent high-affinity transporters found at nerve-terminals and glial cells (Johnston, G. A. R. and Iverson, L. L., J. Neurochem. 18:1951-1961 (1971); Fedele E. and Foster A. C., Brain Res. 572:154-163 (1992)). Two distinct glycine transporters, GlyT1 (Smith, K. E., et al., Neuron 8:927-935 (1992); Liu, Q. R., et al., FEBS Lett. 305:110-114 (1992i); Guastella, J., et al., Proc. Natl. Acad. Sci. (USA) 89:7189-7193 (1992)) and GlyT2 (Liu, Q. R., et al., J. Biol. Chem. 268:22802-22806 (1993)), have been isolated, and share approximately 50% identity at both the nucleotide and amino acid levels. Localization of GlyT1 and GlyT2 by in situ hybridization techniques reveals distinct patterns of expression in the CNS (Liu, Q. R., et al., J. Biol. Chem. 268:22802-22806 (1993); Zafra, F., et al., J. Neurosci. 15:3952-3969 (1995)). GlyT1 is expressed in the hippocampal and cortical regions of the brain, as well as in the spinal cord and brainstem regions. In contrast, GlyT2 is expressed primarily in the spinal cord and cerebellum, and is absent in the hippocampal and cortical regions. Based on their patterns of expression, GlyT1 is thought to co-localize with NMDA receptors, while GlyT2 expression mimics that of strychnine-sensitive glycine receptors (Jursky and Nelson, J. Neurochem. 67:336-344 (1996); Liu, Q. R., et al., J. Biol. Chem. 268:22802-22806 (1993)).
The GlyT1 sequence has been determined for a number of species, including rat (Guastella, J., et al., Proc. Natl. Acad. Sci. (USA) 89:7189-7193 (1992)), mouse (Liu, Q. R., et al., FEBS Lett. 305:110-114 (1992a)) and human (Kim, K-M., et al., Mol. Pharm. 45:608-17 (1994)). Three alternatively spliced forms of the human transporter have been identified (GlyT1a-c) which differ in their amino-terminal sequences (Guastella, J., et al., Proc. Natl. Acad. Sci. (USA) 89:7189-7193 (1992); Liu, Q. R., et al., FEBS Lett. 305:110-114 (1992a); Liu, Q. R., et al., J. Biol. Chem. 268:22802-22806 (1993); Smith, K. E., et al., Neuron 8:927-935 (1992); Borowsky, B., et al., Neuron 10:851-863 (1993); Kim, K-M., et al., Mol. Pharm. 45:608-17 (1994)). The rat GlyT2 sequence has been published (Liu, Q. R., et al., J. Biol. Chem. 268:22802-22806 (1993)), and it also exhibits alternatively spliced forms (GlyT2a and GlyT2b) (Ponce, J., et al., Neurosci. Lett. 242:25-28 (1998)). Recently, two full-length clones described as pHGT2-a and pHGT2-b of human GlyT2 have been constructed (WO98/07854; PCT/US97/14637).
The precise regulation of synaptic glycine concentrations in the CNS is a very important process because glycine is involved in both excitatory and inhibitory neurotransmission (Betz, H., et al., Ann. N. Y. Acad. Sci., 207:109-115 (1993); Benveniste, M., et al., J. Physiol. (London) 428:333-357 (1990)). Glycine transporters are likely to be critical to this process. Compounds able to modulate glycine transporter function (i.e., that inhibit or activate glycine transporter) would be expected to provide a wide variety of therapeutic benefits. For example, glycine receptor inhibition is known to result in pain transmission (Yaksh, Pain, 111-123, (1989)). Therefore, compounds that inhibit GlyT2 transporter activity may increase the activity of neurons having strychnine-sensitive glycine receptors via increasing synaptic levels of glycine, thus diminishing the transmission of pain-related (i.e., nociceptive) information in the spinal cord, which has been shown to be mediated by these receptors. Further, because glycine receptor malfunction is known to play a role in muscle spasticity (Becker, FASEB J. 4:2767-2775 (1990)), compounds that inhibit GlyT2 transporter activity and lead to enhanced inhibitory glycinergic transmission through strychnine-sensitive glycine receptors in the spinal cord can be used to decrease muscle hyperactivity. Such compounds are useful in treating diseases or conditions associated with increased muscle contraction, such as muscle spasticity, myoclonus (which relates to rapid muscle spasms) and epilepsy. Spasticity that may be treated via modulation of glycine receptors is associated with epilepsy, stroke, head trauma, multiple sclerosis, spinal cord injury, dystonia, and other conditions of illness and injury of the nervous system.
The present invention provides novel human glycine transporter type 2 (GlyT2) molecules. Nucleic acids are provided comprising nucleic acid sequences that encode proteins that have glycine transport activity and that have one or more of the following amino acids: (1) serine at a position corresponding to amino acid 24, (2) tryptophan at a position corresponding to amino acid 74, (3) glycine at a position corresponding to amino acid 155, (4) aspartic acid at a position corresponding to amino acid 188, (5) leucine at a position corresponding to amino acid 362, (6) alanine at a position corresponding to amino acid 431, or (7) serine at a position corresponding to amino acid 582, of SEQ ID NO:124. A preferred GlyT2 sequence comprises the amino acid sequence of SEQ ID NO:120. The present invention provides nucleic acids wherein the nucleic acid sequence is that of bases 1 to 2391 of SEQ ID NO: 123 but having one or more of the following nucleotides: (1) A at position 70, (2) T at position 220, (3) G at position 463, (4) G at position 562, (5) T at position 1085, (6) C at position 1292, (7) A at position 1299, (8) T at position 1617, or (9) T at position 1744. A preferred GlyT2 sequence comprises the nucleic acid sequence of SEQ ID NO:119.
Nucleic acids are also provided comprising nucleic acid sequences that encode proteins that have glycine transport activity, that have one or more of the seven amino acids listed above and that additionally have one or more of the following amino acids: (1) leucine at a position corresponding to amino acid 26, (2) leucine at a position corresponding to amino acid 75, (3) valine at a position corresponding to amino acid 89, (4) glycine at a position corresponding to amino acid 102, (5) glutamic acid at a position corresponding to amino acid 174, (6) proline at a position corresponding to amino acid 195, (7) glycine at a position corresponding to amino acid 199, (8) leucine at a position corresponding to amino acid 249, (9) proline at a position corresponding to amino acid 306, (10) glutamic acid at a position corresponding to amino acid 419, (11) asparagine at a position corresponding to amino acid 442, (12) lysine at a position corresponding to amino acid 455, (13) cysteine at a position corresponding to amino acid 458, (14) proline at a position corresponding to amino acid 485, (15) arginine at a position corresponding to amino acid 493, or (16) glutamic acid at a position corresponding to amino acid 650, of SEQ ID NO:124.
The present invention also provides nucleic acids wherein the nucleic acid sequence is that of bases 1 to 2391 of SEQ ID NO:119, 121 or 123 but having one or more of the nine nucleotides listed above but additionally having one or more of the following nucleotides: (a) T at position 77, (b) T at position 220, (c) T at position 244, (d) T at position 266, (e) G at position 304, (f) A at position 521, (g) C at position 583, (h) G at position 596, (i) G at position 678, (j) C at position 681, (k) T at position 745, (1) C at position 750, (m) T at position 765, (n) C or A at position 777, (o) G at position 867, (p) C at position 917, (q) A at position 1256, (r) C at position 1292, (s) A at position 1325, (t) A at position 1364, (u) C at position 1374, (v) A at position 1392, (w) C at position 1454, (x) G at position 1478, (y) C at position 1854, (z) A at position 1949, (aa) C at position 1959, or (bb) C at position 2130.
The present invention also provides nucleic acids comprising nucleic acid sequences that encode proteins that have glycine transport activity and that have one or more of the following amino acids: (1) serine at a position corresponding to amino acid 24, (2) tryptophan at a position corresponding to amino acid 74, (3) glycine at a position corresponding to amino acid 155, (4) aspartic acid at a position corresponding to amino acid 188, (5) leucine at a position corresponding to amino acid 362, (6) alanine at a position corresponding to amino acid 431, or (7) serine at a position corresponding to amino acid 582, of SEQ ID NO:124, and that additionally have one or more of the following amino acids: (1) phenylalanine at a position corresponding to amino acid 124, (2) asparagine at a position corresponding to amino acid 279, (3) glycine at a position corresponding to amino acid 393, (4) asparagine at a position corresponding to amino acid 457, (5) asparagine at a position corresponding to amino acid 463, (6) tyrosine at a position corresponding to amino acid 610, (7) valine at a position corresponding to amino acid 611, (8) serine at a position corresponding to amino acid 733, (9) valine at a position corresponding to amino acid 735, (10) leucine at a position corresponding to amino acid 245, (11) leucine at a position corresponding to amino acid 305, (12) isoleucine at a position corresponding to amino acid 366, or (13) proline at a position corresponding to amino acid 400, of SEQ ID NO: 124.
The present invention also provides nucleic acids wherein the nucleic acid sequence is that of bases 1 to 2391 of SEQ ID NO: 123 but having one or more of the following nucleotides: (1) A at position 70, (2) T at position 220, (3) G at position 463, (4) G at position 562, (5) T at position 1085, (6) C at position 1292, (7) A at position 1299, (8) T at position 1617, or (9) T at position 1744, but additionally having one or more of the following nucleotides: (a) C at position 6, (b) T at position 371, (c) T at position 571, (d) A at position 836, (e) G at position 1116, (f) G at position 1177, (g) C at position 1371, (h) A at position 1387, (i) A at position 1829(j) G at position 1831, (k) C at position 2198, (1) G at position 2203, (m) G at position 342, (n) C at position 733, (o) C at position 913, (p) A at position 951, (q) T at position 1097, (r) C at position 1199, (s) T at position 352, or (t) A at position 2103.
The present invention also provides nucleic acids encoding glycine transporters having at least about 96% sequence identity with the protein sequence of SEQ ID NO: 122 or with a sequence corresponding to the protein sequence of SEQ ID NO: 122 except that it has one or more of the following amino acid substitutions: (1) Pro26 to Leu, (2) Arg74 to Trp, (3) Pro75 to Leu, (4) Ala89 to Val, (5) Ser102 to Gly, (6) Val174 to Glu, (7) Ser195 to Pro, (8) Asp199 to Gly, (9) Val249 to Leu, (10) Leu306 to Pro, (11) Gly419 to Glu, (12) Thr442 to Asn, (13) Thr455 to Lys, (14) Trp458 to Cys, (15) Leu485 to Pro, (16) Lys493 to Arg, or (17) Val650 to Glu. Preferably, the sequence identity is at least about 97%, more preferably at least about 98%, yet more preferably at least about 99%, yet more preferably at least about 99.5%. In an embodiment of the invention, the sequence identity is 100%. Preferably, the encoded glycine transporter has no more than four amino acid differences in the region from amino acid 200 to 797 of reference protein sequence, where the reference sequence is SEQ ID NO: 122 or of a sequence corresponding to the protein sequence of SEQ ID NO:122 except that it has one of the substitutions described above. More preferably, the encoded glycine transporter has no more than two such differences.
The present invention also provides vectors comprising the nucleic acids described above. In one embodiment, the vector is effective to express a glycine transporter mRNA in at least one of a eukaryotic cell or a bacterial cell. In another embodiment of the invention, the vector is effective to express the mRNA in at least one of a mammalian cell, a yeast cell or an avian cell.
The invention further provides an isolated glycine transporter derived from transformed cells according to the present invention, the transporter comprising the amino acid sequence encoded by an above-described nucleic acid or one to two contiguous portions of amino acid sequence encoded by such a nucleic acid, wherein the protein has glycine transporter activity and differs in sequence from the aligned segments of the GlyT2 transporter sequences described by WO98/07854 (PCT/US97/14637) (human) or Liu, et al., J. Biol. Chem. 268:22802-22808 (1993) (rat). Contiguous sequence as used herein, refers to uninterrupted portions of the relevant reference nucleic acid or amino acid sequence. Preferably, the glycine transporter protein of the present invention differs in sequence by at least two amino acids, more preferably, at least four amino acids, from the aligned segments of GlyT2 transporter sequences described by WO98/07854 (PCT/US97/14637) (human) or Liu, et al., J. Biol. Chem. 268:22802-22808 (1993) (rat). Preferably, the contiguous sequences comprise at least about 600 amino acids, more preferably at least about 700 amino acids, more preferably at least about 750 amino acids. In one embodiment, the transporter protein comprises all of the protein sequence encoded by the above-described nucleic acid. Preferably, the transporter protein comprises the amino acid sequence set forth in the protein sequence of SEQ ID NO: 122 or a sequence corresponding to the protein sequence of SEQ ID NO: 122 except that it has one or more of the following amino acid substitutions: (1) Pro26 to Leu, (2) Arg74 to Trp, (3) Pro75 to Leu, (4) Ala89 to Val, (5) Ser102 to Gly, (6) Val174 to Glu, (7) Ser195 to Pro, (8) Asp199 to Gly, (9) Val249 to Leu, (10) Leu306 to Pro, (11) Gly419 to Glu, (12) Thr442 to Asn, (13) Thr455 to Lys, (14) Trp458 to Cys, (15) Leu485 to Pro, (16) Lys493 to Arg, or (17) Val650 to Glu, or an amino acid sequence comprising one to two contiguous portions of these sequences.
The present invention also provides a nucleic acid encoding a transporter protein having at least about 99.5% sequence identity with all or one to two contiguous portions of the amino acid sequence of SEQ ID NO: 122 or with one to two continuous portions of an amino acid sequence corresponding to the protein sequence of SEQ ID NO: 122 except that it has one or more of the following substitutions: (1) Pro26 to Leu, (2) Arg74 to Trp, (3) Pro75 to Leu, (4) Ala89 to Val, (5) Ser102 to Gly, (6) Val174 to Glu, (7) Ser195 to Pro, (8) Asp199 to Gly, (9) Val249 to Leu, (10) Leu306 to Pro, (11) Gly419 to Glu, (12) Thr442 to Asn, (13) Thr455 to Lys, (14) Trp458 to Cys, (15) Leu485 to Pro, (16) Lys493 to Arg, or (17) Val650 to Glu, wherein the encoded protein has glycine transporter activity. Preferably, the contiguous sequences comprise at least about 600 amino acids, more preferably at least about 700 amino acids, more preferably at least about 750 amino acids. The invention also provides a vector comprising this nucleic acid. In one embodiment, the vector is effective to express a glycine transporter mRNA in at least one of a eukaryotic cell or a prokaryotic cell such as a bacterial cell. In another embodiment of the invention, the vector is effective to express the mRNA in at least one of a yeast cell, a mammalian cell or an avian cell.
The present invention additionally provides a cell comprising a first extrinsically-derived nucleic acid according to the first embodiment or a second extrinsically-derived nucleic acid encoding a transporter protein having at least about 99.5% sequence identity with one to two contiguous portions of the protein sequence of SEQ ID NO: 122 or of a sequence corresponding to the protein sequence of SEQ ID NO: 122 except that it has one or more of the following substitutions: (1) Pro26 to Leu, (2) Arg74 to Trp, (3) Pro75 to Leu, (4) Ala89 to Val, (5) Ser102 to Gly, (6) Val174 to Glu, (7) Ser195 to Pro, (8) Asp199 to Gly, (9) Val249 to Leu, (10) Leu306 to Pro, (11) Gly419 to Glu, (12) Thr442 to Asn, (13) Thr455 to Lys, (14) Trp458 to Cys, (15) Leu485to Pro, (16) Lys493 to Arg, or (17) Val650 to Glu, wherein the encoded protein has glycine transporter activity. In one embodiment, the cell expresses a glycine transporter from the nucleic acid. Preferably, the nucleic acid is functionally associated with a promoter that is operative in the cell. In an embodiment of the invention, the promoter is an inducible promoter.
The present invention also provides a method of producing a glycine transporter comprising growing cells as described in the previous paragraphs. This method can further comprise at least one of (a) isolating membranes from said cells, which membranes comprise the glycine transporter or (b) extracting protein fraction from the cells, which fraction comprises the glycine transporter.
The present invention provides a method for characterizing a bioactive agent for treatment of a nervous system disorder or condition or for identifying bioactive agents for treatment of a nervous system disorder or condition, the method comprising (a) providing a first assay composition comprising (i) a cell as described above or (ii) an isolated glycine transporter protein comprising the amino acid sequence encoded by the first or second extrinsically-derived nucleic acids described above, (b) contacting the first assay composition with the bioactive agent or a prospective bioactive agent, and measuring the amount of glycine transport exhibited by the assay composition. Preferably, the method further comprises comparing the amount of glycine transport exhibited by the first assay composition with the amount of glycine transport exhibited by a second such assay composition that is treated the same as the first assay composition except that it is not contacted with the bioactive agent or prospective bioactive agent. The method can be used for characterizing bioactive agents where the nervous system disorder or condition is one of the group including, but not limited to, (a) pain, (b) spasticity, (c) myoclonus, (d) muscle spasm, (e) muscle hyperactivity or (f) epilepsy. In a preferred embodiment, the spasticity for which the bioactive agent is characterized is associated with stroke, head trauma, neuronal cell death, multiple sclerosis, spinal cord injury, dystonia, Huntington""s disease or amyotrophic lateral sclerosis.
The invention further provides a nucleic acid that hybridizes with a reference nucleic acid sequence which is SEQ ID NO:121 or a sequence that varies from the nucleic acid sequence of SEQ ID NO:121 by having one or more of the following substitutions: (a) C77xe2x86x92T, (b) C220xe2x86x92T, (c) C224xe2x86x92T, (d) C266xe2x86x92T, (e) A304xe2x86x92G, (f) T521xe2x86x92A, (g) T583xe2x86x92C, (h) A596xe2x86x92G, (i) A678xe2x86x92G, (j) T681xe2x86x92C, (k) G745xe2x86x92T, (l) G750xe2x86x92C, (m) C765xe2x86x92T, (n) G777xe2x86x92C, (o) A867xe2x86x92G, (p) T917xe2x86x92C, (q) G1256xe2x86x92A, (r) T1292xe2x86x92C, (s) C1325xe2x86x92A, (t) C1364xe2x86x92A, (u) G1374xe2x86x92C, (v) C1392xe2x86x92A, (w) T1454xe2x86x92C, (x) A1478xe2x86x92G, (y) T1854xe2x86x92C, (z) T1949xe2x86x92A, (aa) T1959xe2x86x92C, or (bb) T2130xe2x86x92C, under conditions of sufficient stringency to exclude hybridizations with (a) the sequence for a rat GlyT2 transporter or (b) the sequence for a mammalian GlyT1 transporter. Preferably, the nucleic acid sequence is at least about 18 nucleotides in length and has at least about 95% sequence identity with a sequence embedded in the reference nucleic acid sequence. Preferably the nucleic acid sequence is at least about 40 nucleotides in length, more preferably at least about 100 nucleotides in length. Preferably the nucleic acid sequence has at least about 97% sequence identity with the above-recited reference sequence, more preferably 99% sequence identity. Preferably, the nucleic acid is a PCR primer and the stringent conditions are PCR conditions effective to amplify a human GlyT2 sequence but not amplify (a) the sequence for a rat or mouse GlyT2 transporter or (b) the sequence for a mammalian GlyT1 transporter.
Further, the invention provides a nucleic acid of at least about 18 nucleotides in length comprising a contiguous sequence from the coding or non-coding strand of a human GlyT2 gene or cDNA, wherein the contiguous sequence has at least 1 sequence difference when compared with the rat GlyT2 gene sequence that aligns with the contiguous sequence. Preferably the nucleic acid sequence is at least about 40 nucleotides in length, more preferably at least about 100 nucleotides in length. Preferably, the contiguous sequence has at least two differences, more preferably 3 differences when compared with the rat GlyT2 gene sequence that aligns with the contiguous sequence.
Still further, the invention provides an antisense molecule comprising a contiguous sequence from a coding or non-coding strand of a human gene or cDNA for GlyT2 which is effective when administered to a cell, tissue, organ or animal to reduce the expression of GlyT2 in the cell or in a cell of the tissue, organ or animal, wherein the contiguous sequence has at least 1 sequence difference when compared with the rat GlyT2 gene sequence that aligns with said contiguous sequence. Preferably, the contiguous sequence has at least two differences, more preferably 3 differences when compared with the rat GlyT2 gene sequence that aligns with the contiguous sequence. Antisense molecule is used herein to refer to a molecule designed to bind genomic DNA or mRNA to interfere in transcription or translation, including interfering with mRNA stability. Preferably, the contiguous sequence is at least about 15 nucleotides in length. Preferably, the contiguous stretch is included in the coding or non-coding strand of the nucleic acid sequence of SEQ ID NO:121. The invention further provides an expression vector comprising such an antisense molecule.
The invention also provides a method of reducing GlyT2 expression in a tissue or cell comprising applying to the tissue or cell an amount of such an antisense molecule effective to reduce GlyT2 expression. An amount of an expression vector for expressing such an antisense molecule in a tissue or cell effective to reduce GlyT2 expression is also provided. Alternatively, the invention provides a method of treating a nervous system disorder or condition comprising applying to a tissue or cell of a human patient with a nervous system disorder or condition an amount of such an antisense molecule effective to treat such nervous system disorder or condition or an amount of an expression vector for expressing such an antisense molecule in a tissue or cell effective to treat such nervous system disorder or condition.
Further, the invention provides a method for detecting whether an animal has autoimmune antibodies against a glycine transporter, the method comprising contacting an antibody preparation from the animal or a body fluid from the animal with a polypeptide antigen comprising a glycine transporter or derived from the glycine transporter. Preferably, the polypeptide antigen comprises a contiguous sequence encoded by the protein sequence of SEQ ID NO:122 or with a sequence corresponding to the protein sequence of SEQ ID NO: 122 except that it has one or more of the following substitutions: (1) Pro26 to Leu, (2) Arg74 to Trp, (3) Pro75 to Leu, (4) Ala89 to Val, (5) Ser102 to Gly, (6) Val174 to Glu, (7) Ser195 to Pro, (8) Asp199 to Gly, (9) Val249 to Leu, (10) Leu306 to Pro, (11) Gly419 to Glu, (12) Thr442 to Asn, (13) Thr455 to Lys (14) Trp458 to Cys, (15) Leu485 to Pro, (16) Lys493 to Arg, or (17) Val650 to Glu. Preferably, the contiguous sequence is at least about six amino acids in length, more preferably at least about ten amino acids in length, still more preferably at least about fifteen amino acids in length. In one embodiment of the invention, the peptide antigen is selective for antibodies against either a GlyT1 transporter or a GlyT2 transporter.
Still further, the invention provides the use of a GlyT2 transporter protein to generate GlyT2-specific antibodies.